Factors affecting exocellular polysaccharide production by Lactobacillus delbrueckii subsp. bulgaricus grown in a chemically defined medium

We developed a chemically defined medium (CDM) containing lactose or glucose as the carbon source that supports growth and exopolysaccharide (EPS) production of two strains of Lactobacillus delbrueckii subsp. bulgaricus. The factors found to affect EPS production in this medium were oxygen, pH, temperature, and medium constituents, such as orotic acid and the carbon source. EPS production was greatest during the stationary phase. Composition analysis of EPS isolated at different growth phases and produced under different fermentation conditions (varying carbon source or pH) revealed that the component sugars were the same. The EPS from strain L. delbrueckii subsp. bulgaricus CNRZ 1187 contained galactose and glucose, and that of strain L. delbrueckii subsp. bulgaricus CNRZ 416 contained galactose, glucose, and rhamnose. However, the relative proportions of the individual monosaccharides differed, suggesting that repeating unit structures can vary according to specific medium alterations. Under pH-controlled fermentation conditions, L. delbrueckii subsp. bulgaricus strains produced as much EPS in the CDM as in milk. Furthermore, the relative proportions of individual monosaccharides of EPS produced in pH-controlled CDM or in milk were very similar. The CDM we developed may be a useful model and an alternative to milk in studies of EPS production.     

Influence of end-products inhibition and nutrient limitations on the growth of Lactococcus lactis subsp. lactis

Lactococcus lactis was grown in a simple synthetic medium with glucose as substrate, enabling the precise quantification of each nutrient's contribution to growth. As expected, for the growth of lactic acid bacteria, the growth rate decreased progressively during the cultivation after a short period of exponential growth. End-products of fermentation, predominantly lactate and in minor amounts formate, acetate and ethanol, accumulated within the medium. Growth of the bacterium in fresh media supplemented with these end-products showed that the concentrations attained in the fermentor had no significant influence on the growth rate. As regards nutrients, vitamins and magnesium were never limiting during the culture. On the other hand, amino acid concentrations decreased, some of them being totally consumed and exhausted from the medium before growth ceased. However, growth in reconstituted media constructed with the amino acid concentrations remaining at different times of cultivation showed that amino acid depletion could not account for the observed growth decrease. Batch culture supernatant fluid was used as cultivation medium. Growth rates observed in supernatant cultures supplemented with various nutrients, compared to non-supplemented supernatant, showed that no addition improved growth. Finally, it was concluded that in the experimental conditions used in this study, growth inhibition was predominantly due to phenomena other than lactate inhibition and nutritional limitations, and hence associated with unidentified compounds produced in the fermentation.     

Use of whey for production of exocellular polysaccharide by a mutant strain ofXanthomonas campestris

Growth and kinetics of the production of exocellular polysaccharide was studied in a mutant strain ofXanthomonas campestris lac ⁺ during cultivation in a submerged culture in a medium containing whey. The maximum production of the polymer was observed at the initial stage of the stationary growth phase of the culture. The mean production yield was about 1.4%. The results were comparable with those obtained during cultivation on a lactose medium. Translated by Č. Novotny     

Physiological study of Lactobacillus delbrueckii subsp. bulgaricus strains in a novel chemically defined medium

We developed a chemically defined medium called milieu proche du lait (MPL), in which 22 Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) strains exhibited growth rates ranging from 0.55 to 1 h(-1). MPL can also be used for cultivation of other lactobacilli and Streptococcus thermophilus. The growth characteristics of L. bulgaricus in MPL containing different carbon sources were determined, including an initial characterization of the phosphotransferase system transporters involved. For the 22 tested strains, growth on lactose was faster than on glucose, mannose, and fructose. Lactose concentrations below 0.4% were limiting for growth. We isolated 2-deoxyglucose-resistant mutants from strains CNRZ397 and ATCC 11842. CNRZ397-derived mutants were all deficient for glucose, fructose, and mannose utilization, indicating that these three sugars are probably transported via a unique mannose-specific-enzyme-II-like transporter. In contrast, mutants of ATCC 11842 exhibited diverse phenotypes, suggesting that multiple transporters may exist in that strain. We also developed a protein labeling method and verified that exopolysaccharide production and phage infection can occur in MPL. The MPL medium should thus be useful in conducting physiological studies of L. bulgaricus and other lactic acid bacteria under well controlled nutritional conditions.     

Dialysis Continuous Process for Ammonium-Lactate Fermentation: Improved Mathematical Model and Use of Deproteinized Whey

Separate terms for substrate limitation and product inhibition were incorporated into an equation describing the rate of cell growth for the steady-state fermentation of lactose to lactic acid with neutralization to a constant pH by ammonia. The equation was incorporated into a generalized mathematical model of a dialysis continuous process for the fermentation, developed previously, in which the substrate is fed into the fermentor and the fermentor contents are dialyzed through a membrane against water. The improved model was used to simulate the fermentation on a digital computer, and the results agreed with previous experimental tests using whole whey as the substrate. Further simulations were then made to guide experimental tests using deproteinized whey as the substrate. Dried cheese-whey ultrafiltrate was rehydrated with tap water to contain 242 mg of lactose per ml, supplemented with 8 mg of yeast extract per ml, charged into a 5-liter fermentor without sterilization, adjusted in pH (5.5) and temperature (44°C), and inoculated with an adapted culture of Lactobacillus bulgaricus. The fermentor and dialysate circuits were connected, and a series of steady-state conditions was managed nonaseptically for 71 days. The fermentation of deproteinized whey relative to whole whey, with both highly concentrated, resulted in similar extents of product accumulation but at a lesser rate.     

Effects of various sugars added to growth and drying media upon thermotolerance and survival throughout storage of freeze-dried Lactobacillus delbrueckii ssp. bulgaricus

The aim of this research effort was to investigate the role of various sugar substrates in the growth medium upon thermotolerance and upon survival during storage after freeze-drying of Lactobacillus bulgaricus. Addition of the sugars tested to the growth medium, and of these and sorbitol to the drying medium (skim milk) was investigated so as to determine whether a relationship exists between growth and drying media, in terms of protection of freeze-dried cells throughout storage. The lowest decrease in viability of L. bulgaricus cells after freeze-drying was obtained when that organism was grown in the presence of mannose. However, L. bulgaricus clearly survived better during storage when cells had been grown in the presence of fructose, lactose or mannose rather than glucose (the standard sugar in the growth medium). A similar effect could not be observed in terms of thermotolerance; in this case, the growth medium supplemented with lactose was found to yield cells bearing the highest heat resistance. Supplementation of the drying medium with glucose, fructose, lactose, mannose or sorbitol led in most cases to enhancement of protection during storage, to a degree that was growth medium-dependent.     

Kinetic analysis of batch and continuous culture of Streptococcus cremoris HP1.

The growth of Streptococcus cremoris on a semidefined medium was studied at initial lactose concentrations of 0.2-5.0% in batch culture, and in lactose-limited chemostat cultures at 0.5% lactose. Kinetic analysis of the batch data, using statisitcal techniques, indicated the importance of lactose limitation and lactic acid inhibition of the growth of S. cremoris. A model for the biomass production, lactose utilization, and lactic acid production in batch culture was proposed. In continuous culture, it was found that steady state populations were maintained at higher dilution rates (D = 0.6-0.7 h-1) than the maximum predicted by batch culture (0.56h-1). No evidence for a selection of fast growing mutants was obtained. Copious growth adhering to the walls of the fermentor (i.e. wall growth) occurred very rapidly at higher dilution rates and this undoubtedly affected steady-state growth and wash-out and, as a consequence, the apparent maximum dilution rate.     

High-concentration cultivation of Lactococcus cremoris in a cell-recycle reactor

High-cell concentration cultivation of Lactococcus cremoris, a homofermentative lactic acid producer, in a cell-recycle fermentor is described. Cross-flow filtration allowing continuous removal of the inhibitory metabollte, the influence of dilution rate on growth was investigated in total or partial cell-recycle cultures. The dependence of growth characteristics on operating conditions was identified and quantified using lactose as the carbon source. Growth kinetics could be described by both lactate removal efficiency and nutrient availability. Based on physiological observations, biomass and lactic acid productivities were predicted in partial cell-recycle cultures.     

Increased production of hydrogen peroxide by Lactobacillus delbrueckii subsp. bulgaricus upon aeration: involvement of an NADH oxidase in oxidative stress

The growth of Lactobacillus delbrueckii subsp. bulgaricus (L. delbrueckii subsp. bulgaricus) on lactose was altered upon aerating the cultures by agitation. Aeration caused the bacteria to enter early into stationary phase, thus reducing markedly the biomass production but without modifying the maximum growth rate. The early entry into stationary phase of aerated cultures was probably related to the accumulation of hydrogen peroxide in the medium. Indeed, the concentration of hydrogen peroxide in aerated cultures was two to three times higher than in unaerated ones. Also, a similar shift from exponential to stationary phase could be induced in unaerated cultures by adding increasing concentrations of hydrogen peroxide. A significant fraction of the hydrogen peroxide produced by L. delbrueckii subsp. bulgaricus originated from the reduction of molecular oxygen by NADH catalyzed by an NADH:H(2)O(2) oxidase. The specific activity of this NADH oxidase was the same in aerated and unaerated cultures, suggesting that the amount of this enzyme was not directly regulated by oxygen. Aeration did not change the homolactic character of lactose fermentation by L. delbrueckii subsp. bulgaricus and most of the NADH was reoxidized by lactate dehydrogenase with pyruvate. This indicated that NADH oxidase had no (or a very small) energetic role and could be involved in eliminating oxygen.     

Influence of an initial addition of lactic acid on growth, acid production and their coupling for batch cultures of Lactobacillus helveticus

Growth and production coupling for L. helveticus growing at constant pH?on a medium largely supplemented with yeast extract and peptons has been examined as a function of initial lactic acid addition: these conditions allowed to avoid side effects of cultivation pH?and nitrogen source deficiency on coupling. Growth of L. helveticus strain milano was reduced by half for a free lactic acid concentration of 0.34?g.l^?1; both growth and production were fully inhibited for a critical end-product concentration of 83?g.l^?1, despite the fact that lactose was still available. The extent of autolysis 8?h after growth ceased was practically independent of initial lactic acid concentration: the same conclusion held for the part played by a growth-associated mechanism in total acid production. During the slowing down phase of growth, production was non-growth-associated: production rate remained close to its maximal value, while growth rate declined.     

Culture conditions for the production of esterase from Lactobacillus casei CL96

Culture conditions in growth and esterase production by a newly isolated Lactobacillus casei CL96 were investigated using a dextrose-free MRS medium supplemented with different sugars in a 2 l fermentor at different pHs (4.0-9.0) and temperatures (20-50°C). The optimal growth was obtained in basal MRS medium containing 1% (w/v) lactose at pH 7.0 and 30°C. The maximal esterase production was obtained intracellularly during the late logarithmic phase, but during the stationary phase, the esterase activity was released in the culture medium. The enzyme activity was maximal at pH 7.0 and 37°C. Among various substrates (C₂-C₁₆) tested, the highest activity was towards C₆ and C₈. Though the enzyme was produced constitutively, the tributylin induced the enzyme production by 2.5 fold. L. casei CL96 esterase was very active at neutral pH and ambient temperature and might be suitable for biotechnological applications in the dairy industry.     

Heterologous expression of lactose- and galactose-utilizing pathways from lactic acid bacteria in Corynebacterium glutamicum for production of lysine in whey

The genetic determinants for lactose utilization from Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 and galactose utilization from Lactococcus lactis subsp. cremoris MG 1363 were heterologously expressed in the lysine-overproducing strain Corynebacterium glutamicum ATCC 21253. The C. glutamicum strains expressing the lactose permease and beta-galactosidase genes of L. delbrueckii subsp. bulgaricus exhibited beta-galactosidase activity in excess of 1000 Miller units/ml of cells and were able to grow in medium in which lactose was the sole carbon source. Similarly, C. glutamicum strains containing the lactococcal aldose-1-epimerase, galactokinase, UDP-glucose-1-P-uridylyltransferase, and UDP-galactose-4-epimerase genes in association with the lactose permease and beta-galactosidase genes exhibited beta-galactosidase levels in excess of 730 Miller units/ml of cells and were able to grow in medium in which galactose was the sole carbon source. When grown in whey-based medium, the engineered C. glutamicum strain produced lysine at concentrations of up to 2 mg/ml, which represented a 10-fold increase over the results obtained with the lactose- and galactose-negative control, C. glutamicum 21253. Despite their increased catabolic flexibility, however, the modified corynebacteria exhibited slower growth rates and plasmid instability.     

Biosynthesis of Bacillus intermedius glutamyl endopeptidase in recombinant Bacillus subtilis cells during the stationary growth phase

We studied the biosynthesis of Bacillus intermedius glutamyl endopeptidase in the recombinant Bacillus subtilis strain AJ73 delta58.21 during the stationary growth phase. We optimized the composition of the culture medium to favor effective enzyme production during the stationary growth phase, and found that the nutritional requirements for glutamyl endopeptidase synthesis were different in the stationary phase and growth retardation phase. Proteinase accumulation was activated by complex organic substrates (casein and gelatin). During final stages of the culture growth, the enzyme production was stimulated by Ca2+, Mn2+, and Co2+ and inhibited by Zn2+, Fe2+, and Cu2+. The synthesis of glutamyl endopeptidase in the late stationary phase was not inhibited by glucose, unlike that in the trophophase during proliferation. We conclude that the regulatory mechanisms of proteinase synthesis during vegetative growth and sporulation are different.     

Enhanced inhibitory effect of lactic acid on growth kinetics of Streptococcus cremoris during nutritional medium limitations

Summary In batch cultures of Streptococcus cremoris growth parameters, especially the specific growth rate and its alteration during time-courses of fermentation, were found to be dependent on the culture conditions, in particular the inoculum size and medium composition. It was demonstrated that growth was subject to two main factors, inhibition by lactic acid and limitation by nutritional compounds, the first effect being strongly dependent on medium composition. The tolerance of the strain towards lactic acid was characterized, and critical values of the end-product were related to the concentration of yeast extract and bactotryptone in the medium. After taking into consideration the inhibitory effect of lactic acid, the level of nutritional limitation accounted for the change in specific growth rate during time courses of the culture.     

Biosynthesis of endochitinase of Serratia marcescens.

The growth of a mutant strain of Serratia marcescens with high chitinase activity and the biosynthesis of endochitinase by this strain were investigated. The study was carried out using semisynthetic culture medium without inducers and culture medium containing colloidal chitin as a sole nitrogen and carbon source, with and without mitomycin C. The mutant strain, unlike the native one, was shown to produce endochitinase and to secrete the enzyme into the medium during the growth on culture medium without the inducers, chitin and mitomycin C. During growth on the medium with chitin the mutant strain differed from the native one with a short lag-phase of growth, the early appearance of endochitinase in the culture liquid and a high level of endochitinase activity. The difference between the strains disappeared after the addition of mitomycin C, an inducer of the cell SOS-response, to the culture medium containing chitin. Specific endochitinase activity of S. marcescens mutant strain grown on various culture media had two maxima, namely at the beginning and at the end of the stationary phase. Mitomycin C increased the specific activity in a second peak of endochitinase activity during the growth of the mutant strain.     

Effect of oxygen fluctuations on recombinant Escherichia coli fermentation

Escherichia coli DH5alpha, carrying the pUC19 plasmid for the lacZ fragment of beta-galactosidase and ampicillin resistance, was grown in a batch fermentor under conditions of fluctuating oxygen supply. A Monte Carlo method was used to control the on/off supply of air to simulate circulation of cells in a large fermentor. Rapid changes in oxygen supply reduced the rates of oxygen uptake the carbon dioxide release and prolonged the active second growth phase in batch culture, compared to growth with continuous aeration. Amplification of the plasmid was observed during the stationary phase when air supplied continuously, but not during the Monte Carlo experiments.     

Batch fermentation of whey ultrafiltrate by Lactobacillus helveticus for lactic acid production

Summary Cheese whey ultrafiltrate (WU) was used as the carbon source for the production of lactic acid by batch fermentation with Lactobacillus helveticus strain milano. The fermentation was conducted in a 400 ml fermentor at an agitation rate of 200 rpm and under conditions of controlled temperature (42° C) and pH. In the whey ultrafiltrate-corn steep liquor (WU-CSL) medium, the optimal pH for fermentation was 5.9. Inoculum propagated in skim milk (SM) medium or in lactose synthetic (LS) medium resulted in the best performance in fermentation (in terms of growth, lactic acid production, lactic acid yield and maximum productivity of lactic acid), as compared to that propagated in glucose synthetic (GS) medium. The yeast extract ultrafiltrate (YEU) used as the nitrogen/growth factor source in the WU medium at 1.5% (w/v) gave the highest maximum productivity of lactic acid of 2.70 g/l-h, as compared to the CSL and the tryptone ultrafiltrate (TU). L. helveticus is more advantageous than Streptococcus thermophilus and Lactobacillus delbrueckii for the production of lactic acid from WU. The L. helveticus process will provide an alternative solution to the phage contamination in dairy industries using Lactobacillus bulgaricus.     

Variation in composition and yield of exopolysaccharides produced by Klebsiella sp. strain K32 and Acinetobacter calcoaceticus BD4.

The exopolysaccharides produced by Klebsiella sp. strain K32 and Acinetobacter calcoaceticus BD4 under different growth conditions have been analyzed for sugar composition. The first use of ion chromatography for the quantitative determination of microbial exopolysaccharide composition is reported. Klebsiella sp. strain K32 produced a polymer composed of rhamnose, galactose, and mannose early in its fermentation. The composition of the polymer varied markedly depending on the growth stage of the organism. Klebsiella sp. strain K32 grown in a fermentor produced a polymer which was rich in mannose during early exponential growth in a complex medium, but in the late stationary phase it did not contain detectable levels of mannose. The rhamnose present in the polymer increased from 12 to 55% over the course of growth, whereas galactose decreased from 63 to 45%. A. calcoaceticus BD4 produced a polymer containing rhamnose, glucose, mannose throughout its growth and stationary phase. Klebsiella sp. strain K32 and A. calcoaceticus BD4 were grown on various carbon sources in shake flasks. The polymer yield and composition from both organisms were found to vary with the carbon source. The exopolysaccharide with the highest mannose composition was obtained by using rhamnose as a carbon source for both organisms. These and other data suggest that regulatory changes caused by growth on different substrates result in either the production of a different distribution of polymers or a change in exopolysaccharide structure.     

Variation in composition and yield of exopolysaccharides produced by Klebsiella sp. strain K32 and Acinetobacter calcoaceticus BD4

The exopolysaccharides produced by Klebsiella sp. strain K32 and Acinetobacter calcoaceticus BD4 under different growth conditions have been analyzed for sugar composition. The first use of ion chromatography for the quantitative determination of microbial exopolysaccharide composition is reported. Klebsiella sp. strain K32 produced a polymer composed of rhamnose, galactose, and mannose early in its fermentation. The composition of the polymer varied markedly depending on the growth stage of the organism. Klebsiella sp. strain K32 grown in a fermentor produced a polymer which was rich in mannose during early exponential growth in a complex medium, but in the late stationary phase it did not contain detectable levels of mannose. The rhamnose present in the polymer increased from 12 to 55% over the course of growth, whereas galactose decreased from 63 to 45%. A. calcoaceticus BD4 produced a polymer containing rhamnose, glucose, mannose throughout its growth and stationary phase. Klebsiella sp. strain K32 and A. calcoaceticus BD4 were grown on various carbon sources in shake flasks. The polymer yield and composition from both organisms were found to vary with the carbon source. The exopolysaccharide with the highest mannose composition was obtained by using rhamnose as a carbon source for both organisms. These and other data suggest that regulatory changes caused by growth on different substrates result in either the production of a different distribution of polymers or a change in exopolysaccharide structure.     

Salt tolerance of lactose-grown Vibrio parahaemolyticus carrying Escherichia coli lac genes

Lac- strains of Vibrio parahaemolyticus were converted to Lac+ on receiving a hybrid plasmid containing the lactose utilization genes of Escherichia coli K-12. A V. parahaemolyticus strain containing this hybrid plasmid exhibited optimal growth rates on glucose and other carbon sources in the presence of 0.2 to 0.4 M NaCl. Growth of the same strain on lactose was inhibited at similar concentrations of NaCl. The altered growth rate responses in lactose medium appeared to be attributable to effects of NaCl on the activity of lactose permease, and possibly on that of beta-galactosidase, rather than on the levels of these enzymes in V. parahaemolyticus cells.